Study On The Mechanism Of The Dirigent Proteins To Catalyze Lignans In Isatis Indigotica

The root of Isatis indigotica shows notable anti-viral efficacy and is used as“Banlangen”in traditional Chinese medicine.The“Banlangen Granules”made by“Banlangen”is broadly used for the treatment and prevention of flu,sore throat and virus infection.Rencent studies indicated that the pharmacological activity of“Banlangen”is attributed to a group of lignan metabolites,in which clemastanin B and lariciresinol-4-β-D-glucopyranoside are two representative compounds with highest anti-viral efficacy.Lignans are a class of derivatives derived from the polymerization of two molecules of phenylpropanoid（C6-C3 monomer）via an electron oxidation coupling reaction in cell wall and generally contain multiple chiral centers.Most lignans are also optically active.clemastanin B and lariciresinol-4-β-D-glucopyranoside are the glycosides of the optically active compound（+）-lariciresinol.Therefore,it is very important to study the mechanism and biological significance of the optical activity of lignans in Isatis indigotica.In this study,from the regio-and stereoselectivity of the electron oxidation coupling reaction,two dirigent proteins（DIR）IiDIR1,Ii DIR2 and pinoresinol reductase 1（IiPLR1）were cloned.Through in vitro enzyme activity experiments and in vivo overexpression of hairy root,it was demonstrated that IiDIR1,IiDIR2,and Ii PLR1 played an important role in the formation of the optical rotation of lignans.Furthermore,an AP2/ERF transcription factor Ii AP2/ERF008involved in the regulation of lignans metabolism was obtained.In vitro EMSA experiments,Y1H experiments and in vivo overexpression of hairy roots proved IiAP2/ERF008 had a direct regulation to IiDIR1 and further activated the lignan metabolism pathway,resulting in more lignan metabolites and higher stress resistance.Through phylogenetic analysis,spatio-temporal expression analysis and subcellular localization analysis,2 dirigent proteins（IiDIR1 and IiDIR2）were screened from 19 members（IiDIR1¹9）in this study.To clarify the catalytic function of the DIR protein,Pichia pastoris was used for recombinant Ii DIR1 and IiDIR2 expression.The protein was purified by nickel agarose affinity chromatography and the specificity of the purified protein was verified by western blotting.Finally,purified proteins with a concentration of 0.3 mg ml^-1（IiDIR1）and 0.5 mg ml^-1（IiDIR2）were obtained.Recombinant IiDIR1 and IiDIR2 both were then characterized as the（-）-pinoresinol-forming dirigent proteins（（-）-DIRs）to control the regio-and stereoselective phenol coupling in vitro.To clarify the biological function of the DIR protein in vivo,IiDIR1 and 2-overexpressed（IiDIR1-OVX,IiDIR2-OVX）hairy roots were used to study the function of Ii DIR1 and IiDIR2and resulted in an increase of lignans.Meanwhile,genes in the biosynthetic pathway of lignans were also increased,which indicated that IiDIR1 and IiDIR2 were very significant for lignans biosynthesis and could activate the entire metabolic pathway.The biosynthesis of lignin was also promoted in overexpressed hairy roots.The ratio between G-and S-lignan was changed in Ii DIR1-OVX haity roots.These results confirmed that IiDIR1 and IiDIR2 could regulated the biosynthesis of lignin in vivo,which is related to the stress tolerance.To elucidate the effect of IiPLR1 on the optical activity of lignans,we used E.coli.for expression to obtain a purified protein with the concentration of 5μg ml^-1.The function in vitro showed that Ii PLR1 catalyzed（+）-pinoresinol and（-）-pinoresinol to（+）-lariciresinol and（-）-lariciresinol separately,and then selectively catalyzed（-）-lariciresinol to generate（+）-secoisolariciresinol.Further in vitro and in vivo experiments demonstrated for the first time that Ii DIR1 and IiDIR2,as（-）-DIR,were involved in the synthesis of（-）-pinoresinol by the oxidative coupling reaction,but also worked with IiPLR1 to promote the accumulations of lariciresinol and secoisolariciresinol.Particularly,IiDIR2 can selectively significantly increase the synthesis of（+）-secoisolariciresinol.For the first time,we have demonstrated that DIR is involved in the biosynthesis of lariciresinol and secoisolariciresinol,in addition to its involvement in the biosynthesis of pinoresinol,expanding our understanding of the function of DIR.Based on the lignans and lignin,which play an important role in the growth of plants under adverse environmental conditions,we conducted different biological and abiotic stress treatments on hairy roots.We detected the expression levels of lignan pathway genes by qPCR technology and found that IiDIR1 and IiDIR2 had a very strong response to abiotic stress（NaCl and H₂O₂）and biological stress（flg 22）.The expression of IiDIR1 and IiDIR2 are in the regulatory networks of various plant hormones,such as ethylene（ET）,methyl jasmonate（MeJA）and abscisic acid（ABA）.Both IiDIR1 and IiDIR2 were up-regulated by ET and MeJA.Abscisic acid inhibited the expression of IiDIR1 but promoted the expression of IiDIR2.we further found that over-expressed hairy roots of IiDIR1 and IiDIR2 had more biomass accumulation and faster growth rates than wild-type hairy roots.This shows that IiDIR1 and Ii DIR2 have important implications for the resistance process of Isatis indigotica by affecting the metabolism of lignans and lignin.To investigate the response mechanism of Ii DIR1 and IiDIR2 to hormones,we analyzed the promoters of IiDIR1 and IiDIR2 and found that the promoter region of Ii DIR1 has a conserved GCC-box,suggesting that IiDIR1 might be regulated by an ethylene-responsive transcription factor（AP2/ERF）.Through transcriptome analysis,a total of 119 Ii AP2/ERF transcription factors were obtained.Through bioinformatics analysis,a transcription factor Ii AP2/ERF008 that potentially regulates IiDIR1 was further screened.The qPCR analysis confirmed that the response pattern of the Ii AP2/ERF008 to stress and phytohormones was identical to that of IiDIR1.In vitro EMSA experiment and Y1H experiment confirmed that Ii ERF008 can stably and efficiently bind the GCC-box of the IiDIR1 promoter.In vivo,over-expressed hairy roots of IiERF008（IiERF008-OVX）were successfully constructed and obtained.In IiERF008 over-expressed hairy roots,the expression of all lignan synthesis genes,including IiDIR1,were significantly up-regulated and the lignan content was significantly increased.Combined with in vitro and in vivo experiments,it is fully demonstrated that Ii AP2/ERF008 could regulate the expression level of IiDIR1 through the cis-acting element GCC-box,and further played a role in regulating lignan metabolism,thus responding to adverse stress.In conclusion,the biological functions of IiDIR1 and IiDIR2 were discussed in this study.Based on its function of（-）-pinoresinol synthesis,it was further found that the DIR protein could synergize with PLR to analyse the synthesis of lariciresinol and secoisolariciresinol,expanding the understanding of the function of DIR protein.It has been found that besides lignan metabolism,DIR also regulated lignin metabolism and the ratio of G/S lignin,which implied an important role in increasing hairy root biomass and resisting external stress.We further elucidated that through Ii AP2/ERF008 transcription factor,IiDIR1 and IiDIR2 were in a regulatory network of multiple hormones and stresses.In this study,hairy root overexpression based on IiDIR1,IiDIR2,and IiAP2/ERF008 was studied for metabolic engineering,which significantly increased the content of active substance precursor（+）-lariciresinol in hairy roots.Therefore,IiDIR1,IiDIR2 and IiAP2/ERF008 are potential target genes to improve lignan content and resistance.